Glicentin is a gastrointestinal peptide hormone composed of 69 amino acids which plays an important role in the physiology of gastrointestines and is used in pharmaceuticals, e.g., antidiabetics as disclosed in EPA 0586812.
For commercial production of a useful protein such as human glicentin, a process has been extensively used, which includes producing a fused protein by gene technology by connecting the desired useful protein with a second peptide or a protein and purifying the resultant second protein followed by removing the second peptide or protein portion. Such a process to express a fused protein is expected to increase a production because of an increased expression, an easier purification, an increased stability in production process and an increased secretory efficiency in comparison with a direct expression of the desired useful protein.
However, it is necessary, when a useful protein prepared by gene technology is used as drugs, foods or the like, to remove methionine at the amino terminus encoded by the translation initiation codon attached to a useful protein, since methionine may bring about an immunogenicity. In case of the production by a direct expression of a useful protein, methionine at the amino terminus sometimes remains unremoved, and therefore a process is often used for practical production of a useful protein to produce a fused protein and to remove methionine by digesting the unnecessary moiety.
In producing a useful protein by this process, however, there is a possibility that a part of the fused protein is degraded. This degradation produces contaminants as by-products which are difficult to remove from the desired product. These occurrences lead to a possible contamination of the fused protein or undegraded contaminants in the desired product, and these inclusions are difficult to be removed since the characters of these inclusions are similar to the desired product. Furthermore, these inclusions may possibly bring about an antigenicity like an additional methionine at the amino terminus when the desired product is used as drugs, foods or the like, and therefore it has been highly required to develop an effective removal method of the amino terminal methionine and these contaminants.
In a commercial production of a human glicentin by such processes as disclosed in Japanese Patent Kokai 4-364199 and Japanese Patent Application Hei 5-160977, the presence of a trace amount of contaminants is anticipated, thus requiring a further purification for use as a drug.
Furthermore, a production of proteins in Escherichia coli by gene technology often digests a part of proteins in E. coli with the protease of E. coli itself and results in an inclusion of digested contaminants in the product. It is necessary to remove contaminants as well as the digested substance in a purification process of the afore-mentioned fused proteins or contaminants produced in a cleavage process of the desired protein from a fused protein. In this case, however, it is difficult to remove contaminants since it resembles the desired protein.
Human glicentin is currently produced as a fused protein and also contains contamination from the cleavage reaction. It is highly desirable to find a purification process suitable for removing these contaminants.
On one hand, an effort to purify the desired peptide by introducing a cysteine residue into a peptide has been carried out in the chemical synthesis of peptides. The process is to add the cysteine-methionine sequence to the amino terminus of the desired peptide and to remove, via affinity chromatography against the added cysteine residue, the peptides of which synthesis was terminated before reaching the desired peptide. Even if this method is applied to the synthesis of human glicentin, pure human glicentin cannot be achieved since methionine remains in human glicentin and such human glicentin is decomposed by cyanogen bromide mediated cleavage. The production of long-chain peptides at an industrial level by this chemical synthesis method is practically impossible due to the cost.